This invention concerns optical systems for imaging multiwell sample plates and the like onto camera devices, for analysis and monitoring of light activity in the wells.
Biomedical samples, typically in multiwell sample plates, can be viewed and measured with a CCD camera using a suitable lens. The lens demagnification can be chosen to match the size of the whole sample plate (eg typically 110 mm xc3x9775 mm), or a part of it, to the CCD. The CCD camera can be either a bare cooled CCD, or an image intensified CCD.
Typically a CCD camera sensor is 1xe2x80x3 (25 mm) square. A demagnification of xcx9c110/25=4.4 is therefore necessary to view a whole sample plate.
In modern biomedical assay chemistries where luminescent or fluorescent light emission occurs at long wavelengths towards the red end of the spectrum (600-700 mm) a bare cooled CCD has a great advantage. The CCD is cooled by Peltier or Cryogenic means to reduce the dark noise of the CCD sufficiently. Special electronics is needed to minimise read-out noise, but very low light levels can then be detected in the presence of low noise. The quantum efficiency of a CCD over most of the visible range is 35-40%. Using a thinned back-illuminated CCD, the efficiency can be as high as 8-90%.
The situation can be contrasted with image intensified CCD cameras, where photons are detected in the photocathode of the image intensifier. The quantum efficiency of typical low-noise photocathodes in the red is relatively poor ( less than 5%)). Where Genl image intensifiers are used, there is also usually shading, ie a fall-off of detection efficiency away from the centre of the field of view. Where Gen2 (microchannel plate) image intensifiers are used, there is also a problem at medium and high light levels, where the tube lifetime becomes limited. Gen3 image intensifiers offer much improved quantum efficiency in the red, but these are to some extent in the development stage, at least where tubes of reasonable diameter (eg 40 mm) are involved, and the noise level can be a problem.
With an image intensified CCD a single detected photon results in a burst of electrons in the CCD, spread over a number of pixels. Centroiding methods have been proposed to achieve sub-pixel spatial resolution (eg of the order of 10 microns) for locating the coordinates of a detected photon which is important in some imaging applications where many tiny light emitting sites are present in the sample, and the imaging process requires the different light emitting sites to be resolved the one from the other.
In general, centroiding methods cannot be used with a bare cooled CCD because a detected photon results in only a single electron in the silicon.
Instead of using a lens for imaging, a fibre optic taper can be employed to image the sample plate onto the CCD. A disadvantage however, is that multiple exposures are required to cover the entire plate.
According to one aspect of the present invention, a sample plate is imaged onto a CCD camera by the optical combination of at least one lens and a fibre-optic taper. A fibre optic taper possess some advantages and the use of a converging lens possesses other advantages. As will be apparent from later description however, the invention is able to achieve more than the sum of these differing advantages.
Preferably, the CCD camera is a bare cooled CCD.
Preferably a shutter or iris is included in the light path between the sample plate and the CCD camera.
Where a single lens is employed, the shutter may be located between the lens and the CCD camera faceplate. Where two lenses are employed the shutter may be located between the two lenses.
In general a multiwell sample plate can be imaged and analysed using a system embodying the invention, using a single exposure (shot), in contrast to the situation where a fibre optic taper is employed without a lens, when two or more exposures (shots) are generally required to form a complete image and analyse the entire sample plate.
A second lens may be incorporated to advantage, and typically an imaging lens is located close to the sample and a field lens is located close to the camera input faceplate.
More especially a system incorporating the invention is highly effective in light gathering. Efficiencies of the order of 3.5% can be envisaged.
Thus, preferably the field lens bends the light rays so as to be normal to the taper, hence minimising any loss of light due to rays entering the taper at angles outside the maximum acceptable angle xcex8=sin1 (NA), where NA is the numerical aperture of the taper, (equal to the magnification), ie 24.6/70.7=0.348 in the example given above.
Moreover, in a preferred arrangement, efficient light gathering is achieved by bringing the sample plate as close as possible to the imaging lens and arranging the lens powers to cause the cone of light entering the fibre optic taper just to fill the numerical aperture (NA) of the taper.
In a first preferred embodiment, the taper is 110 mm diameter with a demagnification of 2.87. Lenses having an aperture of the order of F1.1 or better are preferred.
In general, the imaging lens will be a complex lens consisting of a number of separate lens components.
Preferably the light source is a laser light source.
In a further preferred arrangement a second field lens may be mounted close to the sample plate to select rays generally normal to the plate, even near to the edge of the plate, and thereby minimise parallax effects.
Either or both of the field lenses may be a simple single element lens or to advantage may be a multi-element lens.
In general, the imaging and field lenses will comprise a single, multi-component system possessing the property of telecentricity at object and image ie the ability to select rays that are on average normal to the object and image is parallax free.